Crosslinking of ubiquinone cytochrome c reductase (complex III) with periodate-cleavable bifunctional reagents

Smith, Robert J.; Capaldi, Roderick A.; Muchmore, David C.; Dahlquist, Frederick W.

doi:10.1021/bi00611a007

Cited by 83 publications

(43 citation statements)

References 22 publications

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“…The cross-linking reaction was started by adding directly disulfosuccinimidyl tartrate, a water-soluble, homobifunctional N-hydroxysuccinimidyl ester cross-linker, periodate cleavable, to 2 ml of cytosolic fraction obtained by SLO permeabilization of the cell membrane and containing 250 g of total protein in assay buffer to give a final concentration of 1.5 mg of sulfo DST/ml as in Smith (1978). The specific cross-linked products present in the cytosolic fraction were analyzed after immunoprecipitation with anti-myc antibody by two-dimensional gel electrophoresis as in Smith (1978).…”

Section: Cross-linking Experiments Of Emtc Proteins By Using Sulfo Dsmentioning

confidence: 99%

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Endothelial Transcytotic Machinery Involves Supramolecular Protein–Lipid Complexes

Predescu

Palade

2001

MBoC

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We have demonstrated that the plasmalemmal vesicles (caveolae) of the continuous microvascular endothelium function as transcytotic vesicular carriers for protein molecules Ͼ20 Å and that transcytosis is an N-ethylmaleimide-sensitive factor (NSF)-dependent, N-ethylmaleimide-sensitive process. We have further investigated NSF interactions with endothelial proteins to find out 1) whether a complete set of fusion and targeting proteins is present in the endothelium; 2) whether they are organized in multimolecular complexes as in neurons; and 3) whether the endothelial multimolecular complexes differ from their neuronal counterparts, because of their specialized role in transcytosis. To generate the complexes, we have used myc-NSF, cultured pulmonary endothelial cells, and rat lung cytosol and membrane preparations; to detect them we have applied coimmunoprecipitation with myc antibodies; and to characterize them we have used velocity sedimentation and cross-linking procedures. We have found that both cytosolic and membrane fractions contain complexes that comprise beside soluble NSF attachment proteins and SNAREs (soluble NSF attachment protein receptor), rab 5, dynamin, caveolin, and lipids. By immunogold labeling and negative staining we have detected in these complexes, myc-NSF, syntaxin, dynamin, caveolin, and endogenous NSF. Similar complexes are formed by endogenous NSF. The results indicate that complexes with a distinct protein-lipid composition exist and suggest that they participate in targeting, fusion, and fission of caveolae with the endothelial plasmalemma.

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Section: Cross-linking Experiments Of Emtc Proteins By Using Sulfo Dsmentioning

confidence: 99%

“…The specific cross-linked products present in the cytosolic fraction were analyzed after immunoprecipitation with anti-myc antibody by two-dimensional gel electrophoresis as in Smith (1978).…”

Section: Cross-linking Experiments Of Emtc Proteins By Using Sulfo Dsmentioning

confidence: 99%

Endothelial Transcytotic Machinery Involves Supramolecular Protein–Lipid Complexes

Predescu

Palade

2001

MBoC

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“…LKB Ampholines (pH range, 3.5-10) were used. Densitometry of gels was performed with an LKB 2202 laser densitometer equipped with a recording integrator.The crosslinker analysis used dimethyl suberimidate, dimethyl suberate, and disuccinimidyl tartarate (all from Pierce) according to the procedures of Smith et al (20), except that in all cases STOP protein was in MME buffer with 0.1% 2-mercaptoethanol. Glycerol gradients were run according to published procedures (21).…”

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confidence: 99%

“…Protein concentrations were determined by the method of Bradford (17). NaDodSO4/polyacrylamide gel electrophoresis was performed by using the procedures of Schier-Neiss et al (18), and gels were stained either with Coomassie blue R or by using a silver staining procedure (19 (20), except that in all cases STOP protein was in MME buffer with 0.1% 2-mercaptoethanol. Glycerol gradients were run according to published procedures (21).…”

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confidence: 99%

Purification and assay of a 145-kDa protein (STOP145) with microtubule-stabilizing and motility behavior.

Margolis¹,

Rauch²,

Job³

1986

Proc. Natl. Acad. Sci. U.S.A.

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The capacity of microtubules to disassemble in vitro is profoundly affected by a protein factor designated STOP (stable tubule only polypeptide). Here we report the isolation of STOP protein and confirm that its activity is, as predicted, highly substoichiometric to the tubulin in microtubules. The isolation of the 145-kDa STOP (STOP145) protein has been effected from isolated cold-stable microtubules by two column steps: DEAE ion-exchange and a calmodulin affinity column. To confirm the protein's activity we have produced an antibody against STOP145 and have used the antibody to specifically remove the protein and the activity using an antibody-linked affinity column. We conclude that the STOP14S protein accounts for the observed in vitro stabilization of microtubules.The diverse functions that microtubules perform in cells often require the precise control of assembly equilibrium and of polymer stability (1). These properties are invested in the polymer, we believe, by specialized proteins that selectively associate with microtubules for specific purposes. Among these proteins, STOP (stable tubule only polypeptide) is one of particular interest, since it displays stabilizing and motility behavior on the polymer.STOP induces stability in the polymer by preventing the endwise dissociation of subunits. Microtubules in the presence of STOP are indefinitely stable to low temperature, to millimolar calcium, or to assembly-inhibiting drugs. STOP protein is remarkably potent in effect; we have inferred previously that it induced polymer stability at concentrations highly substoichiometric to tubulin (2). Also embodied in the STOP protein is the capacity to slide on microtubules (3). It is not known at this time which activity, stabilization or sliding, predominates in the cell. It is possible that both aspects of its behavior coordinately act to promote intracellular motility. Recent evidence from our laboratory indicates that STOP protein is present among cold-stable microtubules in the spindle of dividing cells (unpublished observations). We have postulated, in this case, that sliding and stabilization may act coordinately to create the poleward movement of chromosomes in anaphase (4).In the initial work on microtubule cold stability, it was reported that the activity was not intrinsic to tubulin but was due to a protein factor associated substoichiometrically with microtubules (5). We tentatively identified a protein of 145 kDa and a few smaller polypeptides as candidates for STOP protein based on their unique association with cold-stable microtubules (2).Here we report on the purification to near homogeneity of a STOP protein of 145 kDa and confirm its substoichiometric activity. Stable microtubules readily respond to simple metabolic signals. They become labile when exposed to ATP (6)(7)(8) Margolis and Wilson (10) and Asnes and Wilson (11), except that all procedures were carried out in MME buffer. After the third cycle of polymerization, the preparation was cleaned by centrifugation of microtubules ...

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“…In general, the mild, neutral conditions of periodate cleavage are compatible with proteins. This has been illustrated in the past, for example, by its application in the detection of protein-protein interactions (29) and the creation of unliganded MHC class I molecules (30). In this article, we report the chemical proteomics application of diol cleavable linker probes.…”

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confidence: 98%

A Simple and Effective Cleavable Linker for Chemical Proteomics Applications

Yang

Hahne

Küster

et al. 2013

Molecular & Cellular Proteomics

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The study of metabolically labeled or probe-modified proteins is an important area in chemical proteomics. Isolation and purification of the protein targets is a necessary step before MS identification. The biotin-streptavidin system is widely used in this process, but the harsh denaturing conditions also release natively biotinylated proteins and non-selectively bound proteins. A cleavable linker strategy is a promising approach for solving this problem. Though several cleavable linkers have been developed and tested, an efficient, easily synthesized, and inexpensive cleavable linker is a desirable addition to the proteomics toolbox. Here, we describe the chemical proteomics application of a vicinal diol cleavable linker. Through easy-to-handle chemistry we incorporate this linker into an activity-based probe and a biotin alkyne tag amenable for bioorthogonal ligation. With these reagents, background protein identifications are significantly reduced relative to standard on-bead digestion. Molecular & Cellular

show abstract

Crosslinking of ubiquinone cytochrome c reductase (complex III) with periodate-cleavable bifunctional reagents

Cited by 83 publications

References 22 publications

Endothelial Transcytotic Machinery Involves Supramolecular Protein–Lipid Complexes

Endothelial Transcytotic Machinery Involves Supramolecular Protein–Lipid Complexes

Purification and assay of a 145-kDa protein (STOP145) with microtubule-stabilizing and motility behavior.

A Simple and Effective Cleavable Linker for Chemical Proteomics Applications

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